Characterization of the DNA binding and structural properties of the BRCT region of human replication factor C p140 subunit

被引:26
作者
Kobayashi, M [1 ]
Figaroa, F [1 ]
Meeuwenoord, N [1 ]
Jansen, LET [1 ]
Siegal, G [1 ]
机构
[1] Leiden Univ, Leiden Inst Chem, NL-2300 RA Leiden, Netherlands
关键词
D O I
10.1074/jbc.M511090200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
BRCT domains, present in a large number of proteins that are involved in cell cycle regulation and/or DNA replication or repair, are primarily thought to be involved in protein-protein interactions. The large (p140) subunit of replication factor C contains a sequence of similar to 100 amino acids in the N-terminal region that binds DNA and is distantly related to known BRCT domains. Here we show that residues 375-480, which include 28 amino acids N-terminal to the BRCT domain, are required for 5'-phosphorylated double-stranded DNA binding. NMR chemical shift analysis indicated that the N-terminal extension includes an alpha-helix and confirmed the presence of a conserved BRCT domain. Sequence alignment of the BRCT region in the p140 subunit of replication factor C from various eukaryotes has identified very few absolutely conserved amino acid residues within the core BRCT domain, whereas none were found in sequences immediately N-terminal to the BRCT domain. However, mapping of the limited number of conserved, surface-exposed residues that were found onto a homology model of the BRCT domain, revealed a clustering on one side of the molecular surface. The cluster, as well as a number of amino acids in the N-terminal alpha-helix, were mutagenized to determine the importance for DNA binding. To ensure minimal structural changes because of the introduced mutations, proteins were checked using one-dimensional H-1 NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal alpha-helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.
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页码:4308 / 4317
页数:10
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